The present invention relates generally to transfection and cell fusion, and, more particularly, to an apparatus and method for directing high-voltage currents to a suspension of cells and DNA, usually contained in a cuvette.
In the biotechnology field, it is known to introduce cloned DNA into mammalian and other cells using a high-voltage electrical discharge. This procedure, commonly referred to as "transfection," typically involves creation of a suspension of cells in a phosphate-buffered saline (PBS) solution and addition of cloned DNA. A high-voltage pulse applied to the suspension from a pulse generator causes the cells to take up and express the exogenous DNA. Various pulse generators are available for this purpose.
The incorporated US patent referenced above provides a transfection high-voltage controller capable of directing a high-voltage current to a suspension of cells and DNA. FIG. 1 is a block schematic diagram of one preferred embodiment for a transfection system 10 disclosed therein for supplying voltage and current to a load 20. Transfection system 10 includes a control processor 30, a charging system 32, a trigger feed 34, a trigger 36, a capacitor 40, a first resistor 42, a second resistor 44, a third resistor 46 and a hi-joule switch 50.
Control processor 30 asserts a control signal to charging system 32 to initiate generation of a charging current. Trigger feed 34 operates on a voltage level present at an output port of the charging system. Trigger feed 34 includes a charge storage device to provide trigger 36 with an appropriate trigger energy.
Resistor 42 is connected to the output port of charging system 32 and to a charging node 52. Resistor 44 is connected between node 52 and ground. Resistor 42 limits current flow into node 52 to permit use of a smaller transformer in charging system 32. Capacitor 40 is coupled between node 52 and ground, and hi-joule switch 50 has an input terminal coupled to node 52. Resistor 44 in series with resistor 46 gradually discharges charge stored on capacitor 40. A monitor signal for control processor 30 is generated by resistor 44 and resistor 46.
Hi-joule switch 50 typically includes a semiconductor-controlled rectifier (SCR) (or silicon-controlled rectifier), as is well known. Hi-joule switch 50 provides an output voltage and current at its output terminal in response to an input gate signal. Once triggered, the charge/energy stored in capacitor 40 is directed through hi-joule switch 50 and into load 20 via a load line 53. Load 20 is commonly a grounded cuvette designed to contain cell suspensions, and the like.
Control processor 30 is connected to trigger 36 and will assert a trigger signal to trigger 36. Responsive to the trigger signal, trigger 36 directs the stored trigger energy as the gating signal for hi-joule switch 50. Typically, the gating signal needs to be a higher voltage than the voltage levels conveniently produced by control processor 30.
Transfection system 10 also includes a manual control unit 54 for setting desired voltage levels and for initiating operation. Typically, manual control unit 54 includes two independent, normally open switches, that both must be held closed in order to operate the system. Such a system reduces the risk of electrical shock to the operator.
In operation, control processor 30 asserts the control signal to the charging system when a user activates manual control 54. Charging system 32 charges capacitor 40 to a voltage level above the prescribed voltage level set by control unit 54, without triggering hi-joule switch 50 as determined by the monitor voltage. Control processor 30 deasserts the control signal to charging system 32 and waits for the voltage level at node 53 (as determined from the monitor voltage) to fall to the proper level, discharged through resistor 44 and resistor 46. When the voltage is correct, control processor 30 asserts the trigger signal to trigger 36, which in turn asserts the gating signal for hi-joule switch 50.
Responsive to the gating signal, hi-joule switch 50 directs the charge stored on capacitor 40 to load 20 via load line 53. The output voltage from hi-joule switch 50 is exponentially decaying, as is well known, and is a function of the capacitance of capacitor 40 and the resistance of load 20.
Transfection system 10 is capable of supplying voltages in excess of 3,000 volts and currents in excess of 1,000 amps. Due to the magnitude of the voltages and currents involved, and the requirement that the equipment be frequently manipulated by human operators, it is a continuous objective to improve the associated safety features and procedures.
As described above, transfection system 10 uses a semiconductor-controlled rectifier (SCR) (or silicon-controlled rectifier) hi-joule switch. This type of apparatus remains the only practical device for delivering clean waveforms at over 1000 ampere currents. Adding several SCR cells in series produces a high-voltage switch. The nature of an SCR is to deliver current up to its fusing current. Above this point, the SCR continues to deliver current, but as a dead-short. Once triggered, the SCR will not turn off until an input current drops below its holding current.
Because of the magnitude of the voltages and currents involved, it is desirable to control output current from the SCR after obtaining the desired output current. As described above, it is futile to try to limit output current by turning the SCR off when output current from the SCR reaches a desired level. Also, due to the potential range of currents involved, it is impractical to use current diverters.
While the load resistance will vary, dependent upon many factors, there are times that the effective resistance of the load will be a minimum of about twenty ohms. The maximum voltage is typically about 2500 volts and therefore the maximum current is normally about 125 amperes. Unfortunately, because of the magnitude of the voltages, an arc-over will sometimes occur, reducing the load (cuvette) resistance to effectively zero ohms. The discharge of the transfection current without resistance can damage the equipment, fuse the SCRs and increase risks to personnel operating the equipment.
It is known to include a limit impedance in series with the load in order to limit maximum current and to thereby decrease risks associated with arc-overs. Such a limit impedance would be added between hi-joule switch 50 and load 20 in load line 53. Even though the series impedance is on the order of about 1.5 ohms, when the cuvette resistance is low, the limit impedance can create a significant voltage drop, altering the actual voltage level applied to the load from the desired level stored on capacitor 40.
The transfection apparatus shown in FIG. 1 includes manual switch 54 for manually operating transfection system 10. In conventional systems during manual operations, it is known to require use of two pulse switches as described above. In order to charge capacitor 40, both switches must be pressed and held together. Such a system works to reduce the chance an operator will contact high voltage portions of the equipment during operation, as long as the charge and discharge processes occur without interruption. If an operator stops the operation short of capacitor discharge, then dangerous voltage levels may be present in capacitor 40.
In order to produce accurate results with transfection system 10, a precise charge/energy must be applied to load 20. In order to accurately determine the precise charge/energy stored in capacitor 40, an accurate capacitance value is necessary. Unfortunately, because of the magnitude of the charge/energy that capacitor 40 must store, the only feasible option up to the present for producing long time constants (up to 3.5 seconds) has been to use electrolytic capacitors. It is prohibitively expensive to obtain precision film capacitors of the size required for long time constants in a transfection system. However, the use of long time constants requires maximum voltages of only 500 V which is possible for electrolytics. For high-voltage (2500 V) experiments, the time constant required is shorter (50 MSEC or less) so that precision (.+-.5% or .+-.10%) film capacitors are feasible. As a consequence, conventional long time constant transfection systems use capacitors with a standard .+-.25% tolerance. The state of the art for electrolytic capacitors is about .+-.20%. Such variations and imprecision in capacitance of storage capacitor 40 reduce the effectiveness of conventional transfection systems since different cells may require specific time-energy delivery.
The lack of precision of electrolytic capacitors is also a problem when providing capacitor expansion modules for transfection system 10. When using transfection system 10, fine control of different levels of charge/energy are required for different applications. It is desirable to provide a set of capacitors that are user selectable in order to obtain desired transfection energy profiles.
In some applications, the desired output voltage will be relatively small. While the SCR is particularly effective as a switch for high voltage and current levels, the SCR can be problematic when using low voltages. For example, sometimes the actual load can be about 1000 ohms. A typical holding current for an SCR is about 60 milliamperes. That requires that the voltage at node 53 be greater than about 60 volts. When the voltage at node 53 falls below 60 volts, with a 1000 ohm load, some conventional hi-joule switches will drop out and truncate the output waveform. In practice, it is desirable that operating voltages for an SCR used in the present application exceed 200 volts for best fidelity. Therefore, it is desirable to provide a hi-joule switch that is capable of operating at both high and low voltage levels.